Rat sperm 2B1 antigen (the orthologue of guinea pig sperm PH20) is a plasma membrane-bound glycoprotein that is endoproteolytically cleaved during passage through the epididymis and subsequently migrates from the tail to the acrosomal domain during capacitation. Unlike guinea pig PH20, however, sperm surface 2B1 is insensitive to phosphatidylinositol phospholipase C, nor is it known how endoproteolytic cleavage affects its hyaluronidase activity. In this investigation we have expressed 2B1 cDNA in Chinese hamster ovary cells; we have shown that it contains an internal sequence motif for attachment of a glycosyl phosphatidylinositol (GPI) anchor and that cleavage from a single- into a two-chain molecule causes a significant shift in the optimum pH for hyaluronidase activity. Functionally, these results suggest that 1) 2B1 glycoprotein on rat spermatozoa is attached to the plasma membrane via a GPI anchor and that this is an important factor in its ability to migrate from the tail to the acrosomal domain during capacitation; and 2) endoproteolytic cleavage of 2B1 serves to optimize its hyaluronidase activity immediately before fertilization, thereby facilitating penetration of spermatozoa through the cumulus oophorus.